The present invention relates to reagent compositions for measuring electrolytes such as calcium ions and chloride ions in body fluids, in particular blood and urine. More specifically, the present invention relates to reagent compositions for measuring the electrolytes by utilizing xcex1-amylases.
Since the electrolyte level of the living body in the normal state is closely regulated by the metabolism, measurement of electrolyte level in body fluids is the most common biochemical clinical test for checking the function of the living body and diagnosing various diseases. For example, measurement of calcium ion level in serum is utilized for diagnosis of diseases involving hypocalcemia, such as hypoprotemia, hypophosphataemia, nephritis, nephrosis, vitamin D deficiency, hypoparathyroidism and rickets, and diseases involving hypercalcemia, such as bone tumors, Addison""s disease, chronic pulmonary emphysema, hyperparathyroidism and renal failure. Further, measurement of chlorine ion level in serum is utilized for diagnosis of diseases involving hypochloremia, such as hypotonic dehydration, hyperglucocorticoidinemia and respiratory acidosis, and diseases involving hyperchloremia, such as hypertonic dehydration, renal tubular acidosis and respiratory alkalosis.
Known methods for measuring calcium ions in body fluids include (1) titration, (2) colorimetry, (3) atomic absorptiometry, (4) flame photometry, (5) electrode method, (6) enzyme method, etc.
The titration (1) is a chemical titration using oxalates or chelates. This method is disadvantageous in that it involves troublesome manipulations, causes operator-to-operator variation in measured value, and is incapable of treating a large amount of a sample in a short period of time. The colorimetry (2) utilizing o-CPC (orthocresolphthalein complexone) as the color developer can be performed with a general-purpose automatic analyzer, but this method is disadvantageous in that it involves temperature- or time-dependent variation in absorbance and is subject to the influence of magnesium ions.
The atomic absorptiometry (3) necessitates dilution of the sample and skilled manipulation to perform. The flame photometry (4) has problems with specificity and reproducibility. The electrode method (5) is disadvantageous in that it is subject to the influence of pH and necessitates apparatus that is difficult to maintain in constant conditions.
The enzyme method (6) includes (i) a method using phospholipase D (Japanese Unexamined Patent Publication No. 195297/1987), (ii) a method using calmodulin (Japanese Unexamined Patent Publication No. 36199/1987), (iii) a method using an xcex1-amylase (Japanese Examined Patent Publication No. 87798/1994), etc.
The method (i) encounters difficulties in preparing a uniform substrate and requires much time for the reaction. The method (ii) necessitate 100- to 1000-fold dilution of the sample. On the other hand, the method (iii) is free from the problems of the above methods (1) to (5) and more precise and easier than the enzyme methods (i) and (ii), and thus has been put into practice in the field of clinical testing. The method (iii) is a method for measuring calcium ions in body fluids wherein an inactivated xcex1-amylase is activated by calcium ions to decompose a sugar substrate, and the decomposition product is measured.
Conventional methods for measuring chlorine ions in body fluids include (A) coulometric titration, (B) ion electrode method, (C) colorimetry and (D) enzyme method. The coulometric titration (A) and ion electrode method (B) require a special apparatus, necessitate careful maintenance and control of the equipment, and have poor analysis efficiency. The colorimetry (C) includes a method using mercury thiocyanate and iron nitrate, which, however, produces waste liquid containing cyan or mercury and thus necessitates special waste liquid treatment, hence disadvantageous.
The enzyme method (D) includes (a) a method using an xcex1-amylase (Japanese Unexamined Patent Publications Nos. 176000/1991, 94698/1992, etc.), (b) a method using sarcosine oxydase (Japanese Unexamined Patent Publication No. 187296/1987) and other methods. The method (a) is a method for measuring chlorine ions in body fluids, wherein an inactivated xcex1-amylase is activated by chlorine ions to decompose a sugar substrate, and the decomposition product is measured. This method is superior in precision, handiness and analysis efficiency to the coulometric titration, ion electrode method and colorimetry, and thus has been put into practice in clinical testing.
In recent clinical testing, reagents are chiefly used in a solution form, for meeting the demand for improvement of precision and stability, cost reduction and labor saving. Conventionally, components unstable in a solution form, such as enzymes, are distributed as lyophilized, and are dissolved in the solvent packaged with the components, at the time of use. Recently, however, enzyme components have higher heat resistance, and can be improved in stability by optimizing pH and buffer conditions, and thus reagent compositions capable of being distributed in a solution form are available. The solution-form reagents are remarkably laborsaving in clinical testing, since it saves the manufacturer lyophilizing the reagents and the test operator dissolving the reagents.
As described above, the electrolyte measuring method utilizing an xcex1-amylase is superior to various other conventional methods in handiness and precision. In this method, the xcex1-amylase is usually inactivated in advance, by removing the target ions to be measured (such as calcium or chlorine ions) which are necessary for expression of the xcex1-amylase activity. The reagents used in this method contain a chelating agent which inhibits a blank reaction, serves as an antagonist to control the quantitativity, and masks interfering ions analogous to the target ions. Since such an inactivated xcex1-amylase is unstable in the presence of a chelating agent, the reagents have the problem that they cannot be stored in a solution form for a prolong period of time.
It is known that oligosaccharides such as maltose and xcex1-cyclodextrin, and their mixtures are useful for stabilizing inactivated xcex1-amylases in the presence of a chelating agent (Japanese Unexamined Patent Publication No. 113894/1994). However, the oligosaccharides are insufficient for stabilizing inactivated xcex1-amylases for a long period of time or at room temperature (18 to 37xc2x0 C.) at which the measurement is to be conducted, although they are useful for stabilization for a short period of time (1 to 2 months) at a low temperature (2 to 8xc2x0 C.).
Further, the solution-form reagents may have the problem of admixture of xcex1-amylases derived from outside the sample. For example, if human saliva is accidentally admixed into a solution-form reagent, xcex1-amylases contained in the saliva decompose the substrate, so that the reagent blank increases and the measurement sensitivity reduces, resulting in lowered quantitativity and impaired precision. Foreign matters such as saliva are likely to be admixed at the time of production or use of reagents, and even a trace amount of sweat- or saliva-derived xcex1-amylases, when admixed, affect the performance characteristics of reagents which may be stored in the form of a solution for a long period of time. Accordingly, xcex1-amylases used in the solution-form reagents need to be stabilized.
In order to cope with the problem of admixed xcex1-amylases derived from outside the sample, Japanese Unexamined Patent Publication No. 277096/1994 discloses a method wherein p-nitrophenyl-xcex2-galactosyl-xcex1-maltopentaoside for use as an xcex1-amylase substrate is made into an aqueous solution at a pH of 2.0 to 5.5 for stabilization. This method can stabilize the xcex1-amylase substrate, but is not sufficiently useful since it imposes limitations on the pH values of the reagent solutions.
As discussed above, although solution-form reagents are mainly used in clinical testing, no solution-form reagents have been developed so far which have sufficiently high solution stability to withstand distribution. Thus, the main object of the present invention is to provide reagent compositions for measuring an electrolyte, which enables enzymatic assay excellent in stability, precision and quantitativity.
The present inventors conducted extensive research to achieve the above object, and found that when the measurement is carried out using two reagent compositions wherein a chelating agent which may cause instability of xcex1-amylases, and an inactivated xcex1-amylase capable of being reversibly reactivated by an electrolyte are formulated separately from each other. The reagent compositions have, substantially without using stabilizers, long-term solution stability at low temperatures or at room temperature at which the reagent compositions are to be used. The present inventors further found that the influence of contaminating xcex1-amylases can be suppressed by using a first reagent composition comprising at least (a) an xcex1-amylase substrate and (b) a chelating agent, and a second reagent composition comprising (c) an inactivated xcex1-amylase capable of being reversibly reactivated by an electrolyte, and the influence can be further reduced when the first reagent composition further contains (d) a substance having xcex1-amylase inhibitory activity. The present invention has been accomplished based on these novel findings.
The present invention provides the following reagent compositions:
(1) A combination of reagent compositions for measuring an electrolyte by utilizing an xcex1-amylase, wherein a chelating agent and an inactivated xcex1-amylase capable of being reversibly reactivated by the electrolyte are formulated separately from each other.
(2) The combination according to Item (1) which comprises a first reagent composition comprising (a) an xcex1-amylase substrate and (b) a chelating agent, and a second reagent composition comprising (c) an inactivated xcex1-amylase capable of being reversibly reactivated by the electrolyte.
(3) The combination according to Item (2) wherein the first reagent composition further comprises a substance having xcex1-amylase inhibitory activity.
(4) The combination according to Item (2) wherein the xcex1-amylase substrate is 2-chloro-4-nitrophenyl-4-o-xcex2-D-galactopyranosyl-xcex1-maltoside.
(5) The combination according to Item (3) wherein the substance having xcex1-amylase inhibitory activity is at least one member selected from the group consisting of 5-bromo-5-nitro-1,3-dioxane, 2-chloroacetamide, 2-hydroxypyridine-N-oxide, imidazolidinyl urea, N-methylisothiazolone, 5-chloro-2-methyl-4-isothiazolin-3-one and N-ethylmaleimide.
(6) The combination according to Item (1) wherein the chelating agent is 1,2-bis(o-aminophenoxy)ethane tetraacetic acid.
(7) The combination according to Item (2) wherein the first reagent composition has a pH of 7 or higher and the second reagent composition has a pH of 6 to 7.
(8) The combination according to Item (1) wherein the electrolyte is calcium ions.
(9) The combination according to Item (1) wherein the electrolyte is chlorine ions.